Orthopox viruses, such as the variola (smallpox) and vaccinia viruses, rely on virally encoded proteins to inhibit host antiviral immune responses. In particular, Orthopoxviruses inhibit activation of the innate immune response. Toll-like receptor 3 (TLR3) is a critical innate immune recognition receptor for these viruses. The ligand for TLR3 is dsRNA, a common intermediate in the lifecycle of many viruses, including pox viruses. Inhibition of TLR3-mediated signaling leads to a significant increase in the levels of poxvirus replication in cell culture. Orthopoxviruses produce several proteins that are believed to interfere with TLR-induced signal transduction. For example, N1L is an inhibitor of TLR3-signal transduction that is required for both viral-mediated inhibition of cytokine production and fully virulent infection. In particular, we have shown that N1L functions by inhibiting two critical signaling pathways downstream of TLR3, NF-?B and IRF3 signaling. NF-?B is responsible, generally, for inducing the inflammatory response, whereas IRF3 is required for the induction of an 'antiviral' state, mediated in large part by the production of type I interferons, especially IFNalpha. We plan to use cell-based assays, designed to monitor the signal-inhibitory activity of N1L, to screen for small molecules which can interfere with this important virally- encoded innate immune inhibitor. Specifically, we plan to identify, in a high-throughput screen, compounds that specifically inhibit N1L-mediated inhibition of NF-?B and IRF3 signaling. By identifying molecules which interfere with N1L, we hope to identify lead compounds that can be used to develop therapeutic antiviral agents targeting orthopoxvirus infections. This is especially relevant to our national biodefense program because of the significant threat posed by the intentional release of smallpox or other similar viruses. Inhibitors of smallpox virus and vaccinia replication have public health value by allowing treatment of unvaccinated victims of smallpox, as no agent is approved to treat smallpox. Inhibitors of smallpox would be essential if vaccine-escape mutants of smallpox were disseminated. Finally compounds that inhibit vaccinia virus would theoretically prevent vaccine (vaccinia) side effects. [unreadable] [unreadable] [unreadable] [unreadable]